Glycoproteins with anti-inflammatory properties

ABSTRACT

Glycoproteins having particular sialylation patterns, and methods of making and using such glycoproteins, are described.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of, and claims priorityto, U.S. patent application Ser. No. 14/416,869, titled “Glycoproteinswith Anti-Inflammatory Properties,” filed on Jan. 23, 2015, whichapplication claims the benefit of U.S. Provisional Application Nos.61/676,253, filed Jul. 26, 2012 and 61/768,027, filed Feb. 22, 2013,which are hereby incorporated by reference in their entirety.

BACKGROUND

Therapeutic glycoproteins are an important class of therapeuticbiotechnology products, and therapeutic Fc containing glycoproteins,such as IVIG, Fc-receptor fusions, and antibodies (including murine,chimeric, humanized and human antibodies and fragments thereof) accountfor the majority of therapeutic biologic products.

SUMMARY

The invention encompasses the discovery that Fc-containing glycoproteinscomprising branched glycans that are sialylated on an α1-3 arm of thebranched glycan in the Fc region, e.g., with a NeuAc-α2,6-Gal terminallinkage, exhibit improved anti-inflammatory properties, e.g., relativeto a reference glycoprotein. Accordingly, the present disclosureencompasses such glycoproteins, as well as methods of making and methodsof using such glycoproteins.

In one aspect, the invention features a method of producing apharmaceutical preparation including glycoproteins having an Fc region,wherein the branched glycans on the Fc region are selectively sialylatedon the α1-3 arm at a predetermined level. This method includes:contacting a sialyltransferase enzyme with a preparation includingglycoproteins having an IgG Fc region under conditions suitable forsialylation of a plurality of the branched glycans by the enzyme;measuring the level of branched glycans having a sialic acid on the α1-3arm and/or on the α1-6 arm; processing the preparation into apharmaceutical preparation if the level is equivalent to thepredetermined level; thereby producing a pharmaceutical preparationincluding glycoproteins having an Fc region, wherein the branchedglycans on the Fc region are selectively sialylated on the α1-3 arm at apredetermined level.

In some embodiments, the predetermined level is at least 95% (e.g., atleast 96%, 97%, 98%, 99%, up to and including 100%) of branched glycanshaving a sialic acid on the α1-3 arm. In other embodiments, thepredetermined level is 20-90% (e.g., 20-30%, 25-35%, 30-40%, 35-45%,40-50%, 45-55%, 50-60%, 55-65%, 60-70%, 65-75%, 70-80%, 75-85%, 80-90%)of branched glycans having a sialic acid on the α1-3 arm.

In certain embodiments, the sialyltransferase enzyme is a ST6Gal-Ienzyme.

In further embodiments, the α1,3 arm of the branched glycans aresialylated with a NeuAc-α2,6-Gal terminal linkage.

In another aspect, the invention features a method of increasinganti-inflammatory activity of a reference glycoprotein preparation. Thismethod includes: providing a reference glycoprotein preparationincluding glycoproteins having an IgG Fc region; and sialylating thebranched glycans on the Fc region on the α1-3 arm of a plurality of thebranched glycans to produce a sialylated glycoprotein preparation;wherein the glycoproteins in the reference glycoprotein preparation arenot IgG glycoproteins or do not consist essentially of an Fc regionderived from IgG glycoproteins; and wherein the sialylated glycoproteinpreparation has an increased level of anti-inflammatory activityrelative to the level of anti-inflammatory activity of the referenceglycoprotein preparation.

In some embodiments, the method further includes measuring in thesialylated glycoprotein preparation the level of the branched glycanshaving a sialic acid on the α1-3 arm and/or measuring the level of thebranched glycans having a sialic acid on the α1-6 arm. In otherembodiments, the method further includes processing the sialylatedglycoprotein preparation into a pharmaceutical preparation if the levelof branched glycans having a sialic aad on the α1-3 arm and/or the levelof branched glycans having a sialic acid on the α1-6 arm meets apredetermined level (e.g., at least about 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of thebranched glycans having a sialic acid on the α1,3 arm and/or less thanabout 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or less of the branchedglycans having a sialic acid on the α1,6 arm).

In another aspect, the invention features a method of increasinganti-inflammatory activity of a reference glycoprotein preparation. Thismethod includes: providing a reference glycoprotein preparationincluding glycoproteins having an IgG Fc region; and sialylating thebranched glycans on the Fc region on the α1-3 arm of a plurality of thebranched glycans to produce a sialylated glycoprotein preparation;measuring in the sialylated glycoprotein preparation the level of thebranched glycans having a sialic acid on the α1-3 arm and/or measuringthe level of the branched glycans having a sialic acid on the α1-6 arm;and processing the sialylated glycoprotein preparation into apharmaceutical preparation if the level of branched glycans having asialic acid on the α1-3 arm and/or the level of branched glycans havinga sialic acid on the α1-6 arm meets a predetermined level; wherein thesialylated glycoprotein preparation has an increased level ofanti-inflammatory activity relative to the level of anti-inflammatoryactivity of the reference glycoprotein preparation.

In some embodiments, the predetermined level of branched glycans havinga sialic acid on the α1-3 arm is at least 95% (e.g., at least 96%, 97%,98%, 99%, up to and including 100%) and said predetermined level ofbranched glycans having a sialic acid on the α1-6 arm is less than 5%.In other embodiments, the predetermined level of branched glycans havinga sialic acid on the α1-3 arm is between 20-90% (e.g., 20-30%, 25-35%,30-40%, 35-45%, 40-50%, 45-55%, 50-60%, 55-65%, 60-70%, 65-75%, 70-80%,75-85%, 80-90%).

In some embodiments, the sialylated glycoprotein preparation has a levelof anti-inflammatory activity that is at least about 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 175%, 200%, 300%, 400%, 500%,or more, higher than the level of anti-inflammatory activity of thereference glycoprotein preparation.

In another aspect, the invention features a method of manufacturing apharmaceutical product including glycoproteins having an IgG Fc region.This method includes: providing a preparation including glycoproteinshaving an IgG Fc region; measuring the level of branched glycans on theFc region in the preparation having a sialic acid on the α1-3 arm and/oron the α1-6 arm; and processing the preparation into a pharmaceuticalproduct if the level of the branched glycans having a sialic acid on theα1-3 arm and/or on the α1-6 arm is equivalent to a predetermined level,thereby manufacturing a pharmaceutical product including glycoproteinshaving an IgG Fc region.

In some embodiments, the predetermined level is a pharmaceuticalspecification of greater than 25% (e.g., greater than 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%,98%, 99%, up to and including 100%) branched glycans having a sialicacid on the α1-3 arm and/or less than 40% (e.g., less than 40%, 35%,30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%) branched glycans having asialic acid on the α1-6 arm.

In other embodiments, the method further includes measuring (e.g., invivo or in vitro) an anti-inflammatory activity of the preparation.

In some embodiments of any of the foregoing methods, the preparation isa preparation of antibodies.

In other embodiments of any of the foregoing methods, the preparation isformulated for intravenous or subcutaneous administration.

In certain embodiments of any of the foregoing methods, theglycoproteins are present in the preparation at a concentration of50-250 mg/mL.

In further embodiments, the glycoproteins consist essentially of an Fcregion.

In other embodiments of any of the foregoing methods, the glycoproteinsfurther have a Fab region.

In some embodiments of any of the foregoing methods, the glycoproteinsare derived from plasma.

In certain embodiments of any of the foregoing methods, theglycoproteins are recombinant glycoproteins.

In further embodiments, the glycoproteins are IgG glycoproteins or saidglycoproteins consist essentially of an Fc region derived from IgGglycoproteins.

In another aspect, the invention features a pharmaceutical preparationincluding sialylated glycoproteins produced by any of the foregoingmethods.

In another aspect, the invention features a pharmaceutical preparationincluding glycoproteins having an Fc region, wherein at least 95% (e.g.,at least 96%, 97%, 98%, 99%, up to and including 100%) of branchedglycans on the Fc region have a sialic acid on the α1-3 arm and do nothave a sialic acid on the α1-6 arm, and wherein the pharmaceuticalpreparation has anti-inflammatory activity.

In another aspect, the invention features a pharmaceutical preparationincluding glycoproteins having an Fc region, wherein 20-90% (e.g.,20-30%, 25-35%, 30-40%, 35-45%, 40-50%, 45-55%, 50-60%, 55-65%, 60-70%,65-75%, 70-80%, 75-85%, 80-90%) of branched glycans on the Fc regionhave a sialic acid on the α1-3 arm and do not have a sialic acid on theα1-6 arm, and wherein the pharmaceutical preparation hasanti-inflammatory activity.

In another aspect, the invention features a pharmaceutical preparationincluding a plurality of glycoproteins having an IgG Fc region, whereinthe IgG Fc region of each of the plurality of glycoproteins includes afirst branched glycan sialylated on the α1-3 arm, and wherein thepharmaceutical preparation has anti-inflammatory activity.

In some embodiments, the IgG Fc region of the plurality of glycoproteinsfurther comprises a second branched glycan. In other embodiments, theIgG Fc region of the plurality of glycoproteins further comprises a highmannose glycan. In certain embodiments, the IgG Fc region of theplurality of glycoproteins further comprises a second branched glycansialylated on the α1-3 arm. In further embodiments, the IgG Fc region ofthe plurality of glycoproteins further comprises a second branchedglycan sialylated on the α1-6 arm.

In some embodiments, the plurality of glycoproteins having an IgG Fcregion includes at least about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the glycoproteins inthe pharmaceutical preparation.

In certain embodiments of any of the foregoing pharmaceuticalpreparations, the pharmaceutical preparation has a level ofanti-inflammatory activity that is at least about 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 175%, 200%, 300%, 400%, 500%,or more, higher than a level of anti-inflammatory activity of areference glycoprotein preparation.

In other embodiments of any of the foregoing pharmaceuticalpreparations, the pharmaceutical preparation is a preparation ofantibodies.

In some embodiments of any of the foregoing pharmaceutical preparations,the pharmaceutical preparation is formulated for subcutaneousadministration.

In certain embodiments of any of the foregoing pharmaceuticalpreparations, the glycoproteins are present in said preparation at aconcentration of 50-250 mg/mL.

In further embodiments of any of the foregoing pharmaceuticalpreparations, the glycoproteins consist essentially of an Fc region.

In other embodiments of any of the foregoing pharmaceuticalpreparations, the glycoproteins further have a Fab region.

In some embodiments of any of the foregoing pharmaceutical preparations,the glycoproteins are derived from plasma.

In certain embodiments of any of the foregoing pharmaceuticalpreparations, the glycoproteins are recombinant glycoproteins.

In further embodiments of any of the foregoing pharmaceuticalpreparations, the glycoproteins are IgG glycoproteins or saidglycoproteins consist essentially of an Fc region derived from IgGglycoproteins.

In some embodiments, the pharmaceutical preparations of the inventionhave increased efficacy in the treatment of rheumatoid arthritis,X-linked agammagloulinemia, hypogammaglobulinemia, an acquiredcompromised immunity condition, immune thrombocytopenia, Kawasakidisease, allogeniec bone marrow transplant, chronic lymphocyticleukemia, common variable immunodeficiency, pediatric HIV, a primaryimmunodeficiency, chronic inflammatory demyelinating polyneuropathy,adult HIV, Alzhemier's disease, autism, Behcet's disease, capillary leaksyndrome, chronic fatigue syndrome, clostridium difficile colitis,dermatomyositis and polymyositis, Grave's ophthalmopathy, musculardystrophy, inclusion body myositis, infertility, Lambert-Eaton syndrome,Lennox-Gastaut, Lupus erythematosus, multifocal motor neuropathy,multiple sclerosis, myasthenia gravis, neonatal alloimmunethrombocytopenia, parvovirus B19, pemphigus, post-transfusion purpura,renal transplant rejection, spontaneous abortion/miscarriage, Sjogren'ssyndrome, stiff person syndrome, opsoclonus myoclonus, severe sepsis andseptic shock, toxic epidermal necrolysis, multiple myeloma, Wegener'sgranulomatosis, Churg-Strauss syndrome, and acute infections relative toIgG (e.g., IVIG).

In some aspects, the present disclosure encompasses a preparation, e.g.,a therapeutic preparation, that includes Fc-containing sialylatedglycoproteins. In some aspects, a preparation, e.g., a therapeuticpreparation, includes a mixture of asialylated glycoproteins,monosialylated glycoproteins (e.g., monosialylated on an α1-3 arm of abranched glycan (e.g., with a NeuAc-α2,6-Gal terminal linkage), and/ordisialylated glycoproteins (e.g., sialylated on both an α1-3 arm and anα1-6 arm of a branched glycan). In some embodiments, a preparation ofglycoproteins includes at least about 75%, at least about 80%, at leastabout 85%, at least about 90%, at least about 95%, or about 100%glycoproteins that are monosialylated on an α3 arm of a branched glycan(e.g., with a NeuAc-α2,6-Gal terminal linkage). In some embodiments, apreparation of glycoproteins includes less than about 25%, less thanabout 20%, less than about 15%, less than about 10%, less than about 5%,or less, asialylated and/or disialylated glycoproteins. In someembodiments, an Fc-containing glycoprotein preparation is selected froma preparation of Fc fragments, a preparation of antibody molecules, apreparation of Fc-fusion proteins (e.g., Fc-receptor fusion proteins),and a preparation of IVIG.

BRIEF DESCRIPTION OF THE DRAWINGS

The present teachings described herein will be more fully understoodfrom the following description of various illustrative embodiments, whenread together with the accompanying drawings. It should be understoodthat the drawings described below are for illustration purposes only andare not intended to limit the scope of the present teachings in any way.

FIGS. 1A-1C show exemplary ST6Gal sialyltransferase sequences. FIG. 1Adepicts an exemplary ST6Gal sialyltransferase amino acid sequence (SEQID NO:1). FIG. 1B depicts an exemplary ST6Gal sialyltransferase aminoacid sequence (SEQ ID NO:2). FIG. 1C depicts an exemplary ST6 Galsialyltransferase amino acid sequence (SEQ ID NO:3).

FIG. 2 is a schematic illustration of a common core pentasaccharide(Man)₃(GlcNAc)(GlcNAc) of N-glycans.

FIG. 3 is a schematic illustration of an IgG antibody molecule.

FIG. 4 a panel of representations of HILIC-LC extracted ion chromatogramof Fc glycopeptides expressed in CHO cells, glycopeptides derived from asialylated Fc that was sialylated using a rhST6Gal expressed in E. colicells, or glycopeptides derived from a sialylated Fc that was sialylatedusing a rhST6Gal expressed in CHO cells.

DETAILED DESCRIPTION

Antibodies are glycosylated at conserved positions in the constantregions of their heavy chain. For example, IgG antibodies have a singleN-linked glycosylation site at Asn297 of the CH2 domain. Each antibodyisotype has a distinct variety of N-linked carbohydrate structures inthe constant regions. For human IgG, the core oligosaccharide normallyconsists of GlcNAc₂Man₃GlcNAc, with differing numbers of outer residues.Variation among individual IgGs can occur via attachment of galactoseand/or galactose-sialic acid at one or both terminal GlcNAc or viaattachment of a third GlcNAc arm (bisecting GlcNAc).

The inventors have discovered that glycoproteins having branched glycansthat are preferentially sialylated on an α1,3 arm of the branched glycanin the Fc region (e.g., with a NeuAc-α2,6-Gal terminal linkage), haveincreased anti-inflammatory properties. Described herein areglycoproteins (e.g., antibodies or fusion proteins, such as Fc fusionproteins) having branched glycans sialylated on an α1,3 arm of thebranched glycan in the Fc region (e.g., with a NeuAc-α2,6-Gal terminallinkage) and have increased anti-inflammatory activity relative toglycoproteins not having such sialylated glycans. Methods of making andusing such compositions are also described.

Definitions

As used herein, the terms “approximately” or “about” as applied to oneor more values of interest, refer to a value that is similar to a statedreference value. In some embodiments, the terms “approximately” or“about” refer to a range of values that fall within 25%, 20%, 19%, 18%,17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%,1%, or less of the stated reference value.

As used herein, the term “equivalent” refers to a difference, forexample, the percent of a particular glycoform in a glycoproteinpreparation, that does not result in a difference in biologicalproperties (e.g., potency, binding characteristics, stability, orsusceptibility to degradation) as compared to a target glycoproteinpreparation. In some instances, “equivalent” refers to the alloweddifference of the percent of a particular glycoform in a glycoproteinpreparation in a specification for commercial release of a drug productunder Section 351(k) of the PHS Act.

As used herein, “glycan” is a sugar, which can be monomers or polymersof sugar residues, such as at least three sugars, and can be linear orbranched (e.g., have an α 1,3 arm and an α 1,6 arm). A “glycan” caninclude natural sugar residues (e.g., glucose, N-acetylglucosamine,N-acetyl neuraminic acid, galactose, mannose, fucose, hexose, arabinose,ribose, xylose, etc.) and/or modified sugars (e.g., 2′-fluororibose,2′-deoxyribose, phosphomannose, 6′sulfo N-acetylglucosamine, etc.). Theterm “glycan” includes homo and heteropolymers of sugar residues. Theterm “glycan” also encompasses a glycan component of a glycoconjugate(e.g., of a glycoprotein, glycolipid, proteoglycan, etc.). The term alsoencompasses free glycans, including glycans that have been cleaved orotherwise released from a glycoconjugate.

As used herein, the term “glycoprotein” refers to a protein thatcontains a peptide backbone covalently linked to one or more sugarmoieties (i.e., glycans). The sugar moiety(ies) may be in the form ofmonosaccharides, disaccharides, oligosaccharides, and/orpolysaccharides. The sugar moiety(ies) may comprise a single unbranchedchain of sugar residues or may comprise one or more branched chains.Glycoproteins can contain O-linked sugar moieties and/or N-linked sugarmoieties.

As used herein, the term “glycoprotein preparation” refers to a set ofindividual glycoprotein molecules, each of which comprises a polypeptidehaving a particular amino acid sequence (which amino acid sequenceincludes at least one glycosylation site) and at least one glycancovalently attached to the at least one glycosylation site. Individualmolecules of a particular glycoprotein within a glycoprotein preparationtypically have identical amino acid sequences but may differ in theoccupancy of the at least one glycosylation sites and/or in the identityof the glycans linked to the at least one glycosylation sites. That is,a glycoprotein preparation may contain only a single glycoform of aparticular glycoprotein, but more typically contains a plurality ofglycoforms. Different preparations of the same glycoprotein may differin the identity of glycoforms present (e.g., a glycoform that is presentin one preparation may be absent from another) and/or in the relativeamounts of different glycoforms.

As used herein, the term “pharmaceutical preparation” refers to apreparation that comprises an active pharmaceutical ingredient or “API”in a dosage form suitable for human or veterinary use (e.g., apreparation in which glycoproteins are present at a concentration of atleast 20 mg/mL).

As used herein, the term “pharmaceutical product” refers to a sterilepreparation intended for human or veterinary use, formulated for use ina subject and presented in its finished dosage form (e.g., packaged foradministration).

“Pharmaceutical preparations” and “pharmaceutical products” can beincluded in kits containing the preparation or product and instructionsfor use.

“Pharmaceutical preparations” and “pharmaceutical products” generallyrefer to compositions in which the final predetermined level ofsialylation has been achieved. To that end, “pharmaceuticalpreparations” and “pharmaceutical products” are substantially free ofST6Gal sialyltransferase and/or sialic acid donor (e.g., cytidine5′-monophospho-N-acetyl neuraminic acid) or the byproducts thereof(e.g., cytidine 5′-monophosphate).

“Pharmaceutical preparations” and “pharmaceutical products” aregenerally substantially free of other components of a cell in which theglycoproteins were produced (e.g., the endoplasmic reticulum orcytoplasmic proteins and RNA), if recombinant.

The term “glycoform” is used herein to refer to a particular form of aglycoprotein. That is, when a glycoprotein includes a particularpolypeptide that has the potential to be linked to different glycans orsets of glycans, then each different version of the glycoprotein (i.e.,where the polypeptide is linked to a particular glycan or set ofglycans) is referred to as a “glycoform”.

“Reference glycoprotein”, as used herein, refers to a glycoproteinhaving substantially the same amino acid sequence as (e.g., having about90-100% identical amino acids of) a glycoprotein described herein, e.g.,a glycoprotein to which it is compared. In some embodiments, a referenceglycoprotein is a therapeutic glycoprotein described herein, e.g., anFDA approved therapeutic glycoprotein.

“Predetermined level,” as used herein, refers to a pre-specifiedparticular level (e.g., an absolute value or range) of one or moreparticular glycans. In some embodiments, a predetermined level is alevel of one or more particular glycans (e.g., branched glycans having asialic acid on an α1-3 arm and/or branched glycans having a sialic acidon an α1-6 arm) in a preparation of a reference glycoprotein. In someembodiments, a predetermined level is expressed as a percent.

For any given parameter, in some embodiments, “percent” refers to thenumber of moles of a particular glycan (glycan X) relative to totalmoles of glycans of a preparation. In some embodiments, “percent” refersto the number of moles of PNGase F-released Fc glycan X relative tototal moles of PNGase F-released Fc glycans detected.

By “purified” (or “isolated”) refers to a nucleic acid sequence (e.g., apolynucleotide) or an amino acid sequence (e.g., a glycoprotein) that isremoved or separated from other components present in its naturalenvironment or substantially free of reactants or byproducts thereofused in its production. For example, a purified or isolated glycoproteinis one that is separated from other components of a cell in which it wasproduced (e.g., the endoplasmic reticulum or cytoplasmic proteins andRNA). A further example of a purified or isolated glycoprotein aresialylated glycoproteins which are substantially free of ST6Galsialyltransferase and/or sialic acid donor (e.g., cytidine5′-monophospho-N-acetyl neuraminic acid) or the byproducts thereof(e.g., cytidine 5′-monophosphate) used in their production. An isolatedpolynucleotide is one that is separated from other nuclear components(e.g., histones) and/or from upstream or downstream nucleic acidsequences. An isolated nucleic acid sequence or amino acid sequence canbe at least 60% free, or at least 75% free, or at least 90% free, or atleast 95% free from other components present in natural environment ofthe indicated nucleic acid sequence or amino acid sequence.

As used herein, “polynucleotide” (or “nucleotide sequence” or “nucleicacid molecule”) refers to an oligonucleotide, nucleotide, orpolynucleotide, and fragments or portions thereof, and to DNA or RNA ofgenomic or synthetic origin, which may be single- or double-stranded,and represent the sense or anti-sense strand.

As used herein, “polypeptide” (or “amino acid sequence” or “protein”)refers to an oligopeptide, peptide, polypeptide, or protein sequence,and fragments or portions thereof, and to naturally occurring orsynthetic molecules. “Amino acid sequence” and like terms, such as“polypeptide” or “protein”, are not meant to limit the indicated aminoacid sequence to the complete, native amino acid sequence associatedwith the recited protein molecule.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

The term “pharmaceutically effective amount” or “therapeuticallyeffective amount” refers to an amount (e.g., dose) effective in treatinga patient, having a disorder or condition described herein. It is alsoto be understood herein that a “pharmaceutically effective amount” maybe interpreted as an amount giving a desired therapeutic effect, eithertaken in one dose or in any dosage or route, taken alone or incombination with other therapeutic agents.

The term “treatment” or “treating”, as used herein, refers toadministering a therapy in an amount, manner, and/or mode effective toimprove a condition, symptom, or parameter associated with a disorder orcondition or to prevent or reduce progression of a disorder orcondition, to a degree detectable to one skilled in the art. Aneffective amount, manner, or mode can vary depending on the subject andmay be tailored to the subject.

The term “subject”, as used herein, means any subject for whomdiagnosis, prognosis, or therapy is desired. For example, a subject canbe a mammal, e.g., a human or non-human primate (such as an ape, monkey,orangutan, or chimpanzee), a dog, cat, guinea pig, rabbit, rat, mouse,horse, cattle, or cow.

As used herein, the term “antibody” refers to a polypeptide thatincludes at least one immunoglobulin variable region, e.g., an aminoacid sequence that provides an immunoglobulin variable domain orimmunoglobulin variable domain sequence. For example, an antibody caninclude a heavy (H) chain variable region (abbreviated herein as VH),and a light (L) chain variable region (abbreviated herein as VL). Inanother example, an antibody includes two heavy (H) chain variableregions and two light (L) chain variable regions. The term “antibody”encompasses antigen-binding fragments of antibodies (e.g., single chainantibodies, Fab, F(ab′)₂, Fd, Fv, and dAb fragments) as well as completeantibodies, e.g., intact immunoglobulins of types IgA, IgG, IgE, IgD,IgM (as well as subtypes thereof). The light chains of theimmunoglobulin can be of types kappa or lambda.

As used herein, the term “constant region” refers to a polypeptide thatcorresponds to, or is derived from, one or more constant regionimmunoglobulin domains of an antibody. A constant region can include anyor all of the following immunoglobulin domains: a CH1 domain, a hingeregion, a CH2 domain, a CH3 domain (derived from an IgA, IgD, IgG, IgE,or IgM), and a CH4 domain (derived from an IgE or IgM).

As used herein, the term “Fc region” refers to a dimer of two “Fcpolypeptides”, each “Fc polypeptide” comprising the constant region ofan antibody excluding the first constant region immunoglobulin domain.In some embodiments, an “Fc region” includes two Fc polypeptides linkedby one or more disulfide bonds, chemical linkers, or peptide linkers.“Fc polypeptide” refers to the last two constant region immunoglobulindomains of IgA, IgD, and IgG, and the last three constant regionimmunoglobulin domains of IgE and IgM, and may also include part or allof the flexible hinge N-terminal to these domains. For IgG, “Fcpolypeptide” comprises immunoglobulin domains Cgamma2 (Cγ2) and Cgamma3(Cγ3) and the lower part of the hinge between Cgamma1 (Cγ1) and Cγ2.Although the boundaries of the Fc polypeptide may vary, the human IgGheavy chain Fc polypeptide is usually defined to comprise residuesstarting at T223 or C226 or P230, to its carboxyl-terminus, wherein thenumbering is according to the EU index as in Kabat et al. (1991, NIHPublication 91-3242, National Technical Information Services,Springfield, Va.). For IgA, Fc polypeptide comprises immunoglobulindomains Calpha2 (Cα2) and Calpha3 (Cα3) and the lower part of the hingebetween Calpha1 (Cα1) and Cα2. An Fc region can be synthetic,recombinant, or generated from natural sources such as IVIG.

An “Fc region-containing glycoprotein” is a glycoprotein that includesall or a substantial portion of an Fc region. Examples of an Fcregion-containing glycoprotein preparation include, e.g., a preparationof Fc fragments, a preparation of antibody molecules, a preparation ofFc-fusion proteins (e.g., an Fc-receptor fusion protein), and apreparation of pooled, polyvalent immunoglobulin molecules (e.g., IVIG).Such an Fc region-containing glycoprotein may be recombinant (e.g., arecombinant Fc fragment preparation or a recombinant antibodypreparation) or naturally derived (such as IVIG).

As used herein, the term “Fc region variant” refers to an analog of anFc region that possesses one or more Fc-mediated activities describedherein. This term includes Fc regions comprising one or more amino acidmodifications (e.g., substitutions, additions, or deletions) relative toa wild type or naturally existing Fc region. For example, variant Fcregions can possess at least about 50% homology, at least about 75%homology, at least about 80% homology, at least about 85%, homology, atleast about 90% homology, at least about 95% homology, or more, with anaturally existing Fc region. For example, variant Fc regions canpossess between 1 and 5 amino acid substitutions, e.g., 1, 2, 3, 4 or 5amino acid substitutions such as phenylalanine to alanine substitutions.Fc region variants also include Fc regions comprising one or more aminoacid residues added to or deleted from the N- or C-terminus of a wildtype Fc region.

As used herein, an “N-glycosylation site of an Fc polypeptide” refers toan amino acid residue within an Fc polypeptide to which a glycan isN-linked. In some embodiments, an Fc region contains a dimer of Fcpolypeptides, and the Fc region comprises two N-glycosylation sites, oneon each Fc polypeptide.

As used herein, the terms “coupled”, “linked”, “joined”, “fused”, and“fusion” are used interchangeably. These terms refer to the joiningtogether of two more elements or components by whatever means, includingchemical conjugation or recombinant means.

The terms “overexpress,” “overexpression,” or “overexpressed”interchangeably refer to a protein or nucleic acid that is transcribedor translated at a detectably greater level, such as in a cancer cell,in comparison to a control cell. The term includes expression due totranscription, post transcriptional processing, translation,post-translational processing, cellular localization (e.g., organelle,cytoplasm, nucleus, cell surface), and RNA and protein stability, ascompared to a control cell. Overexpression can be detected usingconventional techniques, e.g., for detecting mRNA (i.e., RT-PCR, PCR,hybridization) or proteins (i.e., ELISA, immunohistochemicaltechniques). Overexpression can be expression in an amount greater thanabout 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more compared to acontrol cell. In certain instances, overexpression is 1-fold, 2-fold,3-fold, 4-fold, or more, higher level of transcription or translationcompared to a control cell.

As used herein, the term “ST6Gal sialyltransferase” refers to apolypeptide whose amino acid sequence includes at least onecharacteristic sequence of and/or shows at least 100%, 99%, 98%, 97%,96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%,82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71% or 70%identity with a protein involved in transfer of a sialic acid to aterminal galactose of a glycan through an α2,6 linkage (e.g., ST6Gal-I). A wide variety of ST6Gal sialyltransferase sequences are knownin the art. In some embodiments, the ST6Gal sialyltransferase has atleast 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,or 99% identity, or is 100% identical, to amino acid residues 95-416 ofSEQ ID NO:1, to SEQ ID NO:2, or to SEQ ID NO:3 (FIGS. 1A-1C).

I. Glycoproteins

Glycoproteins include, for example, any of a variety of hematologicagents (including, for instance, erythropoietin, blood-clotting factors,etc.), interferons, colony stimulating factors, antibodies, enzymes, andhormones. The identity of a particular glycoprotein is not intended tolimit the present disclosure, and any glycoprotein of interest can be areference glycoprotein in the present methods.

A reference glycoprotein described herein can include a target-bindingdomain that binds to a target of interest (e.g., binds to an antigen).For example, a glycoprotein, such as an antibody, can bind to atransmembrane polypeptide (e.g., receptor) or ligand (e.g., a growthfactor). Exemplary molecular targets (e.g., antigens) for glycoproteinsdescribed herein (e.g., antibodies) include CD proteins such as CD2,CD3, CD4, CD8, CD11, CD19, CD20, CD22, CD25, CD33, CD34, CD40, CD52;members of the ErbB receptor family such as the EGF receptor (EGFR,HER1, ErbB1), HER2 (ErbB2), HER3 (ErbB3) or HER4 (ErbB4) receptor;macrophage receptors such as CRIg; tumor necrosis factors such as TNFαor TRAIL/Apo-2; cell adhesion molecules such as LFA-1, Mac1, p150,95,VLA-4, ICAM-1, VCAM and αvβ3 integrin including either α or β subunitsthereof (e.g., anti-CD11a, anti-CD18 or anti-CD11 b antibodies); growthfactors and receptors such as EGF, FGFR (e.g., FGFR3) and VEGF; IgE;cytokines such as IL1; cytokine receptors such as IL2 receptor; bloodgroup antigens; flk2/flt3 receptor; obesity (OB) receptor; mpl receptor;CTLA-4; protein C; neutropilins; ephrins and receptors; netrins andreceptors; slit and receptors; chemokines and chemookine receptors suchas CCL5, CCR4, CCR5; amyloid beta; complement factors, such ascomplement factor D; lipoproteins, such as oxidized LDL (oxLDL);lymphotoxins, such as lymphotoxin alpha (LTa). Other molecular targetsinclude Tweak, B7RP-1, proprotein convertase subtilisin/kexin type 9(PCSK9), sclerostin, c-kit, Tie-2, c-fms, and anti-M1.

Nonlimiting, exemplary reference glycoproteins that include an Fc regionof an antibody heavy chain include abatacept (Orencia®, Bristol-MyersSquibb), abciximab (ReoPro®, Roche), adalimumab (Humira®, Bristol-MyersSquibb), alefacept (Amevive®, Astellas Pharma), alemtuzumab (Campath®,Genzyme/Bayer), basiliximab (Simulect®, Novartis), bevacizumab(Avastin®, Roche), certolizumab (CIMZIA®, UCB, Brussels, Belgium),cetuximab (Erbitux®, Merck-Serono), daclizumab (Zenapax®, Hoffmann-LaRoche), denileukin diftitox (Ontak®, Eisai), eculizumab (Solids®,Alexion Pharmaceuticals), efalizumab (Raptiva®, Genentech), etanercept(Enbrel®, Amgen-Pfizer), gemtuzumab (Mylotarg®, Pfizer), ibritumomab(Zevalin®, Spectrum Pharmaceuticals), infliximab (Remicade®, Centocor),muromonab (Orthoclone OKT3®, Janssen-Cilag), natalizumab (Tysabri®,Biogen Idec, Elan), omalizumab (Xolair®, Novartis), palivizumab(Synagis®, Medlmmune), panitumumab (Vectibix®, Amgen), ranibizumab(Lucentis®, Genentech), rilonacept (Arcalyst®, RegeneronPharmaceuticals), rituximab (MabThera®, Roche), tositumomab (Bexxar®,GlaxoSmithKline), and trastuzumab (Herceptin®, Roche).

A. N-Linked Glycosylation

N-linked oligosaccharide chains are added to a protein in the lumen ofthe endoplasmic reticulum (see Molecular Biology of the Cell, GarlandPublishing, Inc. (Alberts et al., 1994)). Specifically, an initialoligosaccharide (typically 14-sugar) is added to the amino group on theside chain of an asparagine residue contained within the targetconsensus sequence of Asn-X-Ser/Thr, where X may be any amino acidexcept proline. The structure of this initial oligosaccharide is commonto most eukaryotes, and contains 3 glucose, 9 mannose, and 2N-acetylglucosamine residues. This initial oligosaccharide chain can betrimmed by specific glycosidase enzymes in the endoplasmic reticulum,resulting in a short, branched core oligosaccharide composed of twoN-acetylglucosamine and three mannose residues (depicted in FIG. 2,linked to an asparagine residue). One of the branches is referred to inthe art as the “α1,3 arm”, and the second branch is referred to as the“α1,6 arm”, as denoted in FIG. 2.

N-glycans can be subdivided into three distinct groups called “highmannose type”, “hybrid type”, and “complex type”, with a commonpentasaccharide core (Man(alpha1,6)-(Man(alpha1,3))-Man(beta1,4)-GlcpNAc(beta 1,4)-GlcpNAc(beta1,N)-Asn) occurring in all three groups.

After initial processing in the endoplasmic reticulum, the glycoproteinis transported to the Golgi where further processing may take place. Ifthe glycan is transferred to the Golgi before it is completely trimmedto the core pentasaccharide structure, it results in a “high-mannoseglycan”.

Additionally or alternatively, one or more monosaccharides units ofN-acetylglucosamine may be added to the core mannose subunits to form a“complex glycan”. Galactose may be added to the N-acetylglucosaminesubunits, and sialic acid subunits may be added to the galactosesubunits, resulting in chains that terminate with any of a sialic acid,a galactose or an N-acetylglucosamine residue. Additionally, a fucoseresidue may be added to an N-acetylglucosamine residue of the coreoligosaccharide. Each of these additions is catalyzed by specificglycosyl transferases.

“Hybrid glycans” comprise characteristics of both high-mannose andcomplex glycans. For example, one branch of a hybrid glycan may compriseprimarily or exclusively mannose residues, while another branch maycomprise N-acetylglucosamine, sialic acid, galactose, and/or fucosesugars.

Sialic acids are a family of 9-carbon monosaccharides with heterocyclicring structures. They bear a negative charge via a carboxylic acid groupattached to the ring as well as other chemical decorations includingN-acetyl and N-glycolyl groups. The two main types of sialyl residuesfound in glycoproteins produced in mammalian expression systems areN-acetyl-neuraminic acid (NeuAc) and N-glycolylneuraminic acid (NeuGc).These usually occur as terminal structures attached to galactose (Gal)residues at the non-reducing termini of both N- and O-linked glycans.The glycosidic linkage configurations for these sialyl groups can beeither α2,3 or α2,6.

N-Linked Glycosylation in Antibodies

Antibodies are glycosylated at conserved, N-linked glycosylation sitesin the Fc regions of immunoglobulin heavy chains. For example, eachheavy chain of an IgG antibody has a single N-linked glycosylation siteat Asn297 of the CH2 domain (see Jefferis, Nature Reviews 8:226-234(2009)). IgA antibodies have N-linked glycosylation sites within the CH2and CH3 domains, IgE antibodies have N-linked glycosylation sites withinthe CH3 domain, and IgM antibodies have N-linked glycosylation siteswithin the CH1, CH2, CH3, and CH4 domains (see Arnold et al., J. Biol.Chem. 280:29080-29087 (2005); Mattu et al., J. Biol. Chem. 273:2260-2272(1998); Nettleton et al., Int. Arch. Allergy Immunol. 107:328-329(1995)).

Each antibody isotype has a distinct variety of N-linked carbohydratestructures in the constant regions. For example, IgG has a singleN-linked biantennary carbohydrate at Asn297 of the CH2 domain in each Fcpolypeptide of the Fc region, which also contains the binding sites forC1q and FcγR (see Jefferis et al., Immunol. Rev. 163:59-76 (1998); andWright et al., Trends Biotech 15:26-32 (1997)). For human IgG, the coreoligosaccharide normally consists of GlcNAc₂Man₃GlcNAc, with differingnumbers of outer residues. Variation among individual IgG can occur viaattachment of galactose and/or galactose-sialic acid at one or bothterminal GlcNAc or via attachment of a third GlcNAc arm (bisectingGlcNAc).

B. Antibodies

The basic structure of an IgG antibody is illustrated in FIG. 3. Asshown in FIG. 3, an IgG antibody consists of two identical lightpolypeptide chains and two identical heavy polypeptide chains linkedtogether by disulphide bonds. The first domain located at the aminoterminus of each chain is variable in amino acid sequence, providing theantibody binding specificities found in each individual antibody. Theseare known as variable heavy (VH) and variable light (VL) regions. Theother domains of each chain are relatively invariant in amino acidsequence and are known as constant heavy (CH) and constant light (CL)regions. As shown in FIG. 3, for an IgG antibody, the light chainincludes one variable region (VL) and one constant region (CL). An IgGheavy chain includes a variable region (VH), a first constant region(CH1), a hinge region, a second constant region (CH2), and a thirdconstant region (CH3). In IgE and IgM antibodies, the heavy chainincludes an additional constant region (CH4).

Antibodies described herein can include, for example, monoclonalantibodies, polyclonal antibodies, multispecific antibodies, humanantibodies, humanized antibodies, camelized antibodies, chimericantibodies, single-chain Fvs (scFv), disulfide-linked Fvs (sdFv), andanti-idiotypic (anti-Id) antibodies, and antigen-binding fragments ofany of the above. Antibodies can be of any type (e.g., IgG, IgE, IgM,IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2)or subclass.

The term “Fc fragment”, as used herein, refers to one or more fragmentsof an Fc region that retains an Fc function and/or activity describedherein, such as binding to an Fc receptor. Examples of such fragmentsinclude fragments that include an N-linked glycosylation site of an Fcregion (e.g., an Asn297 of an IgG heavy chain or homologous sites ofother antibody isotypes), such as a CH2 domain. The term “antigenbinding fragment” of an antibody, as used herein, refers to one or morefragments of an antibody that retain the ability to specifically bind toan antigen. Examples of binding fragments encompassed within the term“antigen binding fragment” of an antibody include a Fab fragment, aF(ab′)₂ fragment, a Fd fragment, a Fv fragment, a scFv fragment, a dAbfragment (Ward et al., (1989) Nature 341:544-546), and an isolatedcomplementarity determining region (CDR). These antibody fragments canbe obtained using conventional techniques known to those with skill inthe art, and the fragments can be screened for utility in the samemanner as are intact antibodies.

Reference antibodies or fragments described herein can be produced byany method known in the art for the synthesis of antibodies (see, e.g.,Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring HarborLaboratory Press, 2nd ed. 1988); Brinkman et al., 1995, J. Immunol.Methods 182:41-50; WO 92/22324; WO 98/46645). Chimeric antibodies can beproduced using the methods described in, e.g., Morrison, 1985, Science229:1202, and humanized antibodies by methods described in, e.g., U.S.Pat. No. 6,180,370.

Additional reference antibodies described herein are bispecificantibodies and multivalent antibodies, as described in, e.g., Segal etal., J. Immunol. Methods 248:1-6 (2001); and Tutt et al., J. Immunol.147: 60 (1991).

Naturally derived antibodies that can be used in the methods of theinvention include, for example intravenous immunoglobulin (IVIG) andpolypeptides derived from IVIG (e.g., polypeptides purified from IVIG(e.g., enriched for sialylated IgGs), modified IVIG (e.g., IVIG IgGsenzymatically sialylated), or Fc regions of IVIG (e.g., papain digestedand sialylated)).

IVIG is a blood product containing pooled, polyvalent IgG extracted fromthe plasma of over one thousand blood donors. IVIG is used in thetreatment of rheumatoid arthritis, X-linked agammagloulinemia,hypogammaglobulinemia, an acquired compromised immunity condition,immune thrombocytopenia, Kawasaki disease, allogeniec bone marrowtransplant, chronic lymphocytic leukemia, common variableimmunodeficiency, pediatric HIV, a primary immunodeficiency, chronicinflammatory demyelinating polyneuropathy, adult HIV, Alzhemier'sdisease, autism, Behcet's disease, capillary leak syndrome, chronicfatigue syndrome, clostridium difficile colitis, dermatomyositis andpolymyositis, Grave's ophthalmopathy, muscular dystrophy, inclusion bodymyositis, infertility, Lambert-Eaton syndrome, Lennox-Gastaut, Lupuserythematosus, multifocal motor neuropathy, multiple sclerosis,myasthenia gravis, neonatal alloimmune thrombocytopenia, parvovirus B19,pemphigus, post-transfusion purpura, renal transplant rejection,spontaneous abortion/miscarriage, Sjogren's syndrome, stiff personsyndrome, opsoclonus myoclonus, severe sepsis and septic shock, toxicepidermal necrolysis, multiple myeloma, Wegener's granulomatosis,Churg-Strauss syndrome, and acute infections.

C. Glycoprotein Conjugates

The disclosure includes glycoproteins (or Fc regions or Fc fragmentscontaining one or more N-glycosylation sites thereof) that areconjugated or fused to one or more heterologous moieties and that havedifferent levels of sialylated glycans relative to a correspondingreference glycoprotein. Heterologous moieties include, but are notlimited to, peptides, polypeptides, proteins, fusion proteins, nucleicacid molecules, small molecules, mimetic agents, synthetic drugs,inorganic molecules, and organic molecules. In some instances, areference glycoprotein is a fusion protein that comprises a peptide,polypeptide, protein scaffold, scFv, dsFv, diabody, Tandab, or anantibody mimetic fused to an Fc region, such as a glycosylated Fcregion. The fusion protein can include a linker region connecting the Fcregion to the heterologous moiety (see, e.g., Hallewell et al. (1989),J. Biol. Chem. 264, 5260-5268; Alfthan et al. (1995), Protein Eng. 8,725-731; Robinson & Sauer (1996)).

Exemplary, nonlimiting reference fusion proteins include abatacept(Orencia®, Bristol-Myers Squibb), alefacept (Amevive®, Astellas Pharma),denileukin diftitox (Ontak®, Eisai), etanercept (Enbrel®, Amgen-Pfizer),and rilonacept (Arcalyst®, Regeneron Pharmaceuticals).

In some instances, a reference fusion protein includes an Fc region (oran Fc fragment containing one or more N-glycosylation sites thereof)conjugated to a heterologous polypeptide of at least 10, at least 20, atleast 30, at least 40, at least 50, at least 60, at least 70, at least80, at least 90 or at least 100 amino acids.

In some instances, a reference fusion protein can include an Fc region(or Fc fragment containing one or more N-glycosylation sites thereof)conjugated to marker sequences, such as a peptide to facilitatepurification. A particular marker amino acid sequence is ahexa-histidine peptide, such as the tag provided in a pQE vector(QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311). Otherpeptide tags useful for purification include, but are not limited to,the hemagglutinin “HA” tag, which corresponds to an epitope derived fromthe influenza hemagglutinin protein (Wilson et al., 1984, Cell 37:767)and the “Flag” tag.

In other instances, a reference glycoprotein (or an Fc region or Fcfragment containing one or more N-glycosylation sites thereof) isconjugated to a diagnostic or detectable agent. Such fusion proteins canbe useful for monitoring or prognosing the development or progression ofdisease or disorder as part of a clinical testing procedure, such asdetermining the efficacy of a particular therapy. Such diagnosis anddetection can be accomplished by coupling the glycoprotein to detectablesubstances including, but not limited to, various enzymes, such as butnot limited to horseradish peroxidase, alkaline phosphatase,beta-galactosidase, or acetylcholinesterase; prosthetic groups, such as,but not limited to, streptavidin/biotin and avidin/biotin; fluorescentmaterials, such as, but not limited to, umbelliferone, fluorescein,fluorescein isothiocynate, rhodamine, dichlorotriazinylaminefluorescein, dansyl chloride or phycoerythrin; luminescent materials,such as, but not limited to, luminol; bioluminescent materials, such asbut not limited to, luciferase, luciferin, and aequorin; radioactivematerials, such as but not limited to iodine (¹³¹I, ¹²⁵I, ¹²³I), carbon(¹⁴C), sulfur (³⁵S), tritium (³H), indium (¹¹⁵In, ¹¹³In, ¹¹²In, ¹¹¹In),technetium (⁹⁹Tc), thallium (²⁰¹Ti), gallium (⁶⁸Ga, ⁶⁷Ga), palladium(¹⁰³Pd), molybdenum (⁹⁹Mo), xenon (¹³³Xe), fluorine (¹⁸F), ¹⁵³Sm, ¹⁷⁷Lu,¹⁵³Gd, ¹⁵⁹Gd, ¹⁴⁹Pm, ¹⁴⁰La, ¹⁶⁹Yb, ¹⁷⁵Yb, ¹⁶⁶Ho, ⁹⁰Y, ⁴⁷Sc, ¹⁸⁶Re,¹⁸⁸Re, ¹⁴²Pr, ¹⁰⁵Rh, ⁹⁷Ru, ⁶⁸Ge, ⁵⁷Co, ⁶⁵Zn, ⁸⁵Sr, ³²P, ⁵¹Cr, ⁵⁴Mn,⁷⁵Se, ¹¹³Sn, and ¹¹⁷Sn; positron emitting metals using various positronemission tomographies, non-radioactive paramagnetic metal ions, andmolecules that are radiolabelled or conjugated to specificradioisotopes.

Techniques for conjugating therapeutic moieties to antibodies are wellknown (see, e.g., Arnon et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy”, in Monoclonal AntibodiesAnd Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56. (Alan R. Liss,Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, inControlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53(Marcel Dekker, Inc. 1987)).

II. Sialylated Glycoproteins

Glycoproteins of the present disclosure have glycan compositions thatare different from corresponding reference glycoproteins. For example,the present disclosure encompasses Fc region-containing glycoproteinpreparations (e.g., IVIG, Fc or IgG antibodies) having higher levels ofbranched glycans that are sialylated on an α1-3 arm of the branchedglycans in the Fc region (e.g., with a NeuAc-α2,6-Gal terminal linkage),relative to a corresponding reference IgG antibody. The higher levelscan be measured on an individual Fc region (e.g., an increase in thenumber of branched glycans that are sialylated on an α1-3 arm of thebranched glycans in the Fc region), or the overall composition of apreparation of glycoproteins can be different (e.g., a preparation ofglycoproteins can have a higher number or a higher percentage ofbranched glycans that are sialylated on an α1-3 arm of the branchedglycans in the Fc region) relative to a corresponding preparation ofreference glycoproteins).

In some embodiments, a nucleic acid encoding a reference Fcregion-containing glycoprotein described herein is co-expressed in ahost cell with one or more sialyltransferase enzymes, e.g., an α2,6sialyltransferase (e.g., ST6Gal-I). Sialyltransferase enzymes are knownin the art and are commercially available.

Methods and compositions described herein include the use of asialyltransferase enzyme, e.g., an α2,6 sialyltransferase (e.g.,ST6Gal-I). A number of ST6Gal sialyltransferases are known in the artand are commercially available (see, e.g., Takashima, Biosci.Biotechnol. Biochem. 72:1155-1167 (2008); Weinstein et al., J. Biol.Chem. 262:17735-17743 (1987)). ST6Gal-I catalyzes the transfer of sialicacid from a sialic acid donor (e.g., cytidine 5′-monophospho-N-acetylneuraminic acid) to a terminal galactose residue of glycans through anα2,6 linkage. The sialic acid donor reaction product is cytidine5′-monophosphate.

In some embodiments, the disclosure encompasses methods of modifyingactivity of a sialyltransferase enzyme, e.g., a sialylating activity ofa sialyltransferase enzyme. In some embodiments, activity is modified byexpressing a sialyltransferase enzyme in eukaryotic cells (e.g., yeast,insect, or mammalian cells such as CHO cells), purifying thesialyltransferase, and contacting the sialyltransferase with an Fcregion-containing glycoprotein, thereby preferentially sialylating theα1,3 arms of branched glycans of the Fc region-containing glycoprotein.In some embodiments, such sialylated Fc region-containing glycoproteinsexhibit anti-inflammatory activity.

In some embodiments, activity is modified by expressing asialyltransferase enzyme in prokaryotic cells (e.g., bacterial cells,e.g., E. coli), purifying the sialyltransferase, and contacting thesialyltransferase with an Fc region-containing glycoprotein, therebypreferentially sialylating the α1,6 arms of branched glycans of the Fcregion-containing glycoprotein. In some embodiments, such sialylated Fcregion-containing glycoproteins do not exhibit anti-inflammatoryactivity.

In some embodiments, an Fc region-containing glycoprotein isco-expressed in a host cell with a sialyltransferase enzyme (e.g.,ST6Gal sialyltransferase), and the enzyme sialylates a branched glycanas described herein.

In some embodiments, an Fc region-containing glycoprotein is expressedin a host cell, and the host cell endogenously expresses orrecombinantly expresses a sialyltransferase (e.g., ST6Galsialyltransferase). Additionally or alternatively, the host cell iscultured under conditions that increase the activity of asialyltransferase (e.g., ST6Gal sialyltransferase) in the cell, therebyproducing an Fc region-containing glycoprotein having branched glycanssialylated as described herein.

Recombinant expression of a gene, such as a nucleic acid encoding areference glycoprotein and/or a sialyltransferase described herein, caninclude construction of an expression vector containing a polynucleotidethat encodes a reference polypeptide and/or a sialyltransferase. Once apolynucleotide has been obtained, a vector for the production of thereference polypeptide can be produced by recombinant DNA technologyusing techniques known in the art. Known methods can be used toconstruct expression vectors containing polypeptide coding sequences andappropriate transcriptional and translational control signals. Thesemethods include, for example, in vitro recombinant DNA techniques,synthetic techniques, and in vivo genetic recombination.

An expression vector can be transferred to a host cell by conventionaltechniques, and the transfected cells can then be cultured usingconventional techniques to produce reference polypeptides.

A variety of host expression vector systems can be used (see, e.g., U.S.Pat. No. 5,807,715). Such host-expression systems can be used to producepolypeptides and, where desired, subsequently purified. Such hostexpression systems include microorganisms such as bacteria (e.g., E.coli and B. subtilis) transformed with recombinant bacteriophage DNA,plasmid DNA or cosmid DNA expression vectors containing polypeptidecoding sequences; yeast (e.g., Saccharomyces and Pichia) transformedwith recombinant yeast expression vectors containing polypeptide codingsequences; insect cell systems infected with recombinant virusexpression vectors (e.g., baculovirus) containing polypeptide codingsequences; plant cell systems infected with recombinant virus expressionvectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus,TMV) or transformed with recombinant plasmid expression vectors (e.g. Tiplasmid) containing polypeptide coding sequences; or mammalian cellsystems (e.g., COS, CHO, BHK, 293, NS0, and 3T3 cells) harboringrecombinant expression constructs containing promoters derived from thegenome of mammalian cells (e.g., metallothionein promoter) or frommammalian viruses (e.g., the adenovirus late promoter; the vacciniavirus 7.5K promoter).

For bacterial systems, a number of expression vectors can be used,including, but not limited to, the E. coli expression vector pUR278(Ruther et al., 1983, EMBO 12:1791); pIN vectors (Inouye & Inouye, 1985,Nucleic Acids Res. 13:3101-3109; Van Heeke & Schuster, 1989, J. Biol.Chem. 24:5503-5509); and the like. pGEX vectors can also be used toexpress foreign polypeptides as fusion proteins with glutathione5-transferase (GST).

For expression in mammalian host cells, viral-based expression systemscan be utilized (see, e.g., Logan & Shenk, 1984, Proc. Natl. Acad. Sci.USA 8 1:355-359). The efficiency of expression can be enhanced by theinclusion of appropriate transcription enhancer elements, transcriptionterminators, etc. (see, e.g., Bittner et al., 1987, Methods in Enzymol.153:516-544).

In addition, a host cell strain can be chosen that modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins and gene products. Appropriatecell lines or host systems can be chosen to ensure the correctmodification and processing of the polypeptide expressed. Such cellsinclude, for example, established mammalian cell lines and insect celllines, animal cells, fungal cells, and yeast cells. Mammalian host cellsinclude, but are not limited to, CHO, VERY, BHK, HeLa, COS, MDCK, 293,313, W138, BT483, Hs578T, HTB2, BT20 and T47D, NS0 (a murine myelomacell line that does not endogenously produce any immunoglobulin chains),CRL7O3O and HsS78Bst cells.

For long-term, high-yield production of recombinant proteins, host cellsare engineered to stably express a polypeptide. Host cells can betransformed with DNA controlled by appropriate expression controlelements known in the art, including promoter, enhancer, sequences,transcription terminators, polyadenylation sites, and selectablemarkers. Methods commonly known in the art of recombinant DNA technologycan be used to select a desired recombinant clone.

In some embodiments, a reference Fc region-containing glycoprotein isrecombinantly produced in cells as described herein, purified, andcontacted with a sialyltransferase enzyme in vitro to produce Fcregion-containing glycoproteins containing higher levels of glycanshaving higher levels of sialic acid on the α1-3 arms of the branchedglycans with a NeuAc-α2,6-Gal terminal linkage, relative to thereference glycoprotein. In some embodiments, a purified referenceglycoprotein is contacted with the sialyltransferase in the presence ofCMP-sialic acid, manganese, and/or other divalent metal ions.

A reference Fc region-containing glycoprotein can be purified by anymethod known in the art for purification, for example, by chromatography(e.g., ion exchange, affinity, and sizing column chromatography),centrifugation, differential solubility, or by any other standardtechnique for the purification of proteins. For example, a referenceantibody can be isolated and purified by appropriately selecting andcombining affinity columns such as Protein A column with chromatographycolumns, filtration, ultra filtration, salting-out and dialysisprocedures (see Antibodies: A Laboratory Manual, Ed Harlow, David Lane,Cold Spring Harbor Laboratory, 1988). Further, as described herein, areference glycoprotein can be fused to heterologous polypeptidesequences to facilitate purification.

In accordance with the present disclosure, there may be employedconventional molecular biology, microbiology, and recombinant DNAtechniques within the skill of the art. Such techniques are described inthe literature (see, e.g., Sambrook, Fritsch & Maniatis, MolecularCloning: A Laboratory Manual, Second Edition (1989) Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.; DNA Cloning: A PracticalApproach, Volumes I and II (D. N. Glover ed. 1985); OligonucleotideSynthesis (M. J. Gait ed. 1984); Nucleic Acid Hybridization (B. D. Hames& S. J. Higgins eds. (1985)); Transcription And Translation (B. D. Hames& S. J. Higgins, eds. (1984)); Animal Cell Culture (R. I. Freshney, ed.(1986)); Immobilized Cells and Enzymes (IRL Press, (1986)); B. Perbal, APractical Guide To Molecular Cloning (1984); F. M. Ausubel et al.(eds.), Current Protocols in Molecular Biology, John Wiley & Sons, Inc.(1994).

In some embodiments, a glycoprotein can be purified using a lectincolumn by methods known in the art (see, e.g., WO 02/30954). Forexample, a preparation of glycoproteins can be enriched forglycoproteins containing glycans having sialic acids in α2,6 linkage asdescribed in, e.g., WO2008/057634. Following enrichment of glycoproteinscontaining glycans having sialic acids in α2,6 linkage, the glycancomposition of such glycoproteins can be further characterized toidentify glycoproteins having sialic acids attached to the α1,3 arm of abranched glycan. Preparations of glycoproteins containing apredetermined level of glycans having sialic acids in α2,6 linkage onthe α1,3 arm can be selected for use, e.g., for therapeutic use. Suchcompositions can have increased levels of anti-inflammatory activity.

In some embodiments, a glycoprotein, e.g., a glycosylated antibody, issialylated after the glycoprotein is produced. For example, aglycoprotein can be recombinantly expressed in a host cell (as describedherein) and purified using standard methods. The purified glycoproteinis then contacted with an ST6Gal sialyltransferase (e.g., arecombinantly expressed and purified ST6Gal sialyltransferase) in thepresence of reaction conditions as described herein. In certainembodiments, the conditions include contacting the purified glycoproteinwith an ST6Gal sialyltransferase in the presence of a sialic acid donor,e.g., cytidine 5′-monophospho-N-acetyl neuraminic acid, manganese,and/or other divalent metal ions. In some embodiments, IVIG is used in asialylation method described herein.

In some embodiments, chemoenzymatic sialylation is used to sialylateglycoproteins. Briefly, this method involves sialylation of a purifiedbranched glycan, followed by incorporation of the sialylated branchedglycan en bloc onto a polypeptide to produce a sialylated glycoprotein.

A branched glycan can be synthesized de novo using standard techniquesor can be obtained from a glycoprotein preparation (e.g., a recombinantglycoprotein, Fc, or IVIG) using an appropriate enzyme, such as anendoglycosidase (e.g., EndoH or EndoF). After sialylation of thebranched glycan, the sialylated branched glycan can be conjugated to apolypeptide using an appropriate enzyme, such as a transglycosidase, toproduce a sialylated glycoprotein.

In some embodiments, a branched glycan used in methods described hereinis a galactosylated branched glycan (e.g., includes a terminal galactoseresidue). In some embodiments, a branched glycan is galactosylatedbefore being sialylated using a method described herein. In someembodiments, a branched glycan is first contacted with agalactosyltransferase (e.g., a beta-1,3-galactosyltransferase) andsubsequently contacted with an ST6Gal sialyltransferase as describedherein. In some embodiments, a galactosylated glycan is purified beforebeing contacted with an ST6Gal sialyltransferase. In some embodiments, agalactosylated glycan is not purified before being contacted with anST6Gal sialyltransferase. In some embodiments, a branched glycan iscontacted with a galactosyltransferase and an ST6Gal sialyltransferasein a single step.

Glycan compositions can be characterized using methods described in,e.g., Barb, Biochemistry 48:9705-9707 (2009); Anumula, J. Immunol.Methods 382:167-176 (2012); Gilar et al., Analytical Biochem. 417:80-88(2011).

III. Glycan Evaluation

In some embodiments, glycans of glycoproteins are analyzed by anyavailable suitable method. In some instances, glycan structure andcomposition as described herein are analyzed, for example, by one ormore, enzymatic, chromatographic, mass spectrometry (MS),chromatographic followed by MS, electrophoretic methods, electrophoreticmethods followed by MS, nuclear magnetic resonance (NMR) methods, andcombinations thereof. Exemplary enzymatic methods include contacting aglycoprotein preparation with one or more enzymes under conditions andfor a time sufficient to release one or more glycan(s) (e.g., one ormore exposed glycan(s)). In some instances, the one or more enzymesinclude(s) PNGase F. Exemplary chromatographic methods include, but arenot limited to, Strong Anion Exchange chromatography using PulsedAmperometric Detection (SAX-PAD), liquid chromatography (LC), highperformance liquid chromatography (HPLC), ultra performance liquidchromatography (UPLC), thin layer chromatography (TLC), amide columnchromatography, and combinations thereof. Exemplary mass spectrometry(MS) include, but are not limited to, tandem MS, LC-MS, LC-MS/MS, matrixassisted laser desorption ionisation mass spectrometry (MALDI-MS),Fourier transform mass spectrometry (FTMS), ion mobility separation withmass spectrometry (IMS-MS), electron transfer dissociation (ETD-MS), andcombinations thereof. Exemplary electrophoretic methods include, but arenot limited to, capillary electrophoresis (CE), CE-MS, gelelectrophoresis, agarose gel electrophoresis, acrylamide gelelectrophoresis, SDS-polyacrylamide gel electrophoresis (SDS-PAGE)followed by Western blotting using antibodies that recognize specificglycan structures, and combinations thereof. Exemplary nuclear magneticresonance (NMR) include, but are not limited to, one-dimensional NMR(1D-NMR), two-dimensional NMR (2D-NMR), correlation spectroscopymagnetic-angle spinning NMR (COSY-NMR), total correlated spectroscopyNMR (TOCSY-NMR), heteronuclear single-quantum coherence NMR (HSQC-NMR),heteronuclear multiple quantum coherence (HMQC-NMR), rotational nuclearoverhauser effect spectroscopy NMR (ROESY-NMR), nuclear overhausereffect spectroscopy (NOESY-NMR), and combinations thereof.

In some instances, techniques described herein may be combined with oneor more other technologies for the detection, analysis, and or isolationof glycans or glycoproteins. For example, in certain instances, glycansare analyzed in accordance with the present disclosure using one or moreavailable methods (to give but a few examples, see Anumula, Anal.Biochem., 350(1):1, 2006; Klein et al., Anal. Biochem., 179:162, 1989;and/or Townsend, R. R. Carbohydrate Analysis” High Performance LiquidChromatography and Capillary Electrophoresis, Ed. Z. El Rassi, pp181-209, 1995; WO2008/128216; WO2008/128220; WO2008/128218;WO2008/130926; WO2008/128225; WO2008/130924; WO2008/128221;WO2008/128228; WO2008/128227; WO2008/128230; WO2008/128219;WO2008/128222; WO2010/071817; WO2010/071824; WO2010/085251;WO2011/069056; and WO2011/127322, each of which is incorporated hereinby reference in its entirety). For example, in some instances, glycansare characterized using one or more of chromatographic methods,electrophoretic methods, nuclear magnetic resonance methods, andcombinations thereof.

In some instances, methods for evaluating one or more target proteinspecific parameters, e.g., in a glycoprotein preparation, e.g., one ormore of the parameters disclosed herein, can be performed by one or moreof following methods.

In some instances, methods for evaluating one or more target proteinspecific parameters, e.g., in a glycoprotein preparation, e.g., one ormore of the parameters disclosed herein, can be performed by one or moreof following methods.

TABLE 1 Exemplary methods of evaluating parameters: Method(s) Relevantliterature Parameter C18 UPLC Mass Spec.* Chen and Flynn, Anal.Biochem., Glycan(s) 370: 147-161 (2007) (e.g., N-linked glycan, exposedN- Chen and Flynn, J. Am. Soc. Mass linked glycan, glycan detection,Spectrom., 20: 1821-1833 (2009) glycan identification, andcharacterization; site specific glycation; glycoform detection (e.g.,parameters 1-7); percent glycosylation; and/or aglycosyl) Peptide LC-MSDick et al., Biotechnol. Bioeng., C-terminal lysine(reducing/non-reducing) 100: 1132-1143 (2008) Yan et al., J. Chrom. A.,1164: 153-161 (2007) Chelius et al., Anal. Chem., 78: 2370- 2376 (2006)Miller et al., J. Pharm. Sci., 100: 2543- 2550 (2011) LC-MS(reducing/non- Dick et al., Biotechnol. Bioeng., reducing/alkylated)100: 1132-1143 (2008) Goetze et al., Glycobiol., 21: 949-959 (2011) Weakcation exchange Dick et al., Biotechnol. Bioeng., (WCX) chromatography100: 1132-1143 (2008) LC-MS (reducing/non- Dick et al., Biotechnol.Bioeng., N-terminal pyroglu reducing/alkylated) 100: 1132-1143 (2008)Goetze et al., Glycobiol., 21: 949-959 (2011) PeptideLC-MS Yan et al.,J. Chrom. A., 1164: 153-161 (reducing/non-reducing) (2007) Chelius etal., Anal. Chem., 78: 2370- 2376 (2006) Miller et al., J. Pharm. Sci.,100: 2543- 2550 (2011) Peptide LC-MS Yan et al., J. Chrom. A., 1164:153-161 Methionine oxidation (reducing/non-reducing) (2007); Xie et al.,mAbs, 2: 379-394 (2010) Peptide LC-MS Miller et al., J. Pharm. Sci.,100: 2543- Site specific glycation (reducing/non-reducing) 2550 (2011)Peptide LC-MS Wang et al., Anal. Chem., 83: 3133-3140 Free cysteine(reducing/non-reducing) (2011); Chumsae et al., Anal. Chem., 81: 6449-6457 (2009) Bioanalyzer Forrer et al., Anal. Biochem., 334: 81-88 Glycan(e.g., N-linked glycan, (reducing/non-reducing)* (2004) exposed N-linkedglycan) (including, for example, glycan detection, identification, andcharacterization; site specific glycation; glycoform detection; percentglycosylation; and/or aglycosyl) LC-MS (reducing/non- Dick et al.,Biotechnol. Bioeng., Glycan (e.g., N-linked glycan, reducing/alkylated)*100: 1132-1143 (2008) exposed N-linked glycan) * Methods include Goetzeet al., Glycobiol., 21: 949-959 (including, for example, glycan removal(e.g., enzymatic, (2011) detection, identification, and chemical, andphysical) Xie et al., mAbs, 2: 379-394 (2010) characterization; sitespecific of glycans glycation; glycoform detection; percentglycosylation; and/or aglycosyl) Bioanalyzer Forrer et al., Anal.Biochem., 334: 81-88 Light chain: Heavy chain (reducing/non-reducing)(2004) Peptide LC-MS Yan et al., J. Chrom. A., 1164: 153-161Non-glycosylation-related peptide (reducing/non-reducing) (2007)modifications (including, for Chelius et al., Anal. Chem., 78: 2370-example, sequence analysis and 2376 (2006) identification of sequencevariants; Miller et al., J. Pharm. Sci., 100: 2543- oxidation;succinimide; aspartic 2550 (2011) acid; and/or site-specific asparticacid) Weak cation exchange Dick et al., Biotechnol. Bioeng., Isoforms(including, for example, (WCX) chromatography 100: 1132-1143 (2008)charge variants (acidic variants and basic variants); and/or deamidatedvariants) Anion-exchange Ahn et al., J. Chrom. B, 878: 403-408Sialylated glycan chromatography (2010) Anion-exchange Ahn et al., J.Chrom. B, 878: 403-408 Sulfated glycan chromatography (2010)1,2-diamino-4,5- Hokke et al., FEBS Lett., 275: 9-14 Sialic acidmethylenedioxybenzene (1990) (DMB) labeling method LC-MS Johnson et al.,Anal. Biochem., 360: 75- C-terminal amidation 83 (2007) LC-MS Johnson etal., Anal. Biochem., 360: 75- N-terminal fragmentation 83 (2007)Circular dichroism Harn et al., Current Trends in Secondary structure(including, for spectroscopy Monoclonal Antibody Development andexample, alpha helix content Manufacturing, S. J. Shire et al., eds,and/or beta sheet content) 229-246 (2010) Intrinsic and/or ANS dye Harnet al., Current Trends in Tertiary structure (including, forfluorescence Monoclonal Antibody Development and example, extent ofprotein folding) Manufacturing, S. J. Shire et al., eds, 229-246 (2010)Hydrogen-deuterium Houde et al., Anal. Chem., 81: 2644- Tertiarystructure and dynamics exchange-MS 2651 (2009) (including, for example,accessibility f amide protons to solvent water) Size-exclusion Carpenteret al., J. Pharm. Sci., Extent of aggregation chromatography 99:2200-2208 (2010) Analytical Pekar and Sukumar, Anal. Biochem.,ultracentrifugation 367: 225-237 (2007)The literature recited above are hereby incorporated by reference intheir entirety or, in the alternative, to the extent that they pertainto one or more of the methods for determining a parameter describedherein.

IV. Anti-Inflammatory Properties

The inventors have discovered that sialic acid-mediatedanti-inflammatory properties on Fc-containing molecules are not only dueto the level of sialylation, but due to particular branchingarrangements. Accordingly, Fc region-containing glycoproteins describedherein (e.g., Fc region-containing glycoproteins containing glycanscontaining sialic acid on α1,3 arms of branched glycans with aNeuAc-α2,6-Gal terminal linkage) have increased anti-inflammatoryproperties relative to a reference glycoprotein.

In some embodiments, Fc region-containing glycoproteins containingsialic acid on α1,3 arms of branched glycans with a NeuAc-α2,6-Galterminal linkages exhibit increased anti-inflammatory activity relativeto a reference glycoprotein, e.g., a level of anti-inflammatory activitythat is at least 10%, at least 20%, at least 30%, at least 40%, at least50%, at least 60%, at least 70%, at least 80%, at least 90%, at least100%, at least 125%, at least 150%, at least 175%, at least 200%, atleast 250%, at least 300%, or higher, relative to a referenceglycoprotein.

In some embodiment, Fc region-containing glycoproteins having sialicacids in both the α1,3 and α1,6 arms of branched glycans may inhibitanti-inflammatory activity of Fc-containing glycoproteins.

V. Pharmaceutical Compositions and Administration

A glycoprotein of the present disclosure (e.g., an Fc region-containingglycoprotein comprising branched glycans that are sialylated on an α1,3arm of the branched glycan in the Fc region, e.g., with a NeuAc-α2,6-Galterminal linkage), can be incorporated into a pharmaceutical compositionand can exhibit anti-inflammatory activity. Such a pharmaceuticalcomposition is useful as an improved composition for the preventionand/or treatment of diseases relative to the corresponding referenceglycoprotein. Pharmaceutical compositions comprising a glycoprotein canbe formulated by methods known to those skilled in the art. Thepharmaceutical composition can be administered parenterally in the formof an injectable formulation comprising a sterile solution or suspensionin water or another pharmaceutically acceptable liquid. For example, thepharmaceutical composition can be formulated by suitably combining thesialylated glycoprotein with pharmaceutically acceptable vehicles ormedia, such as sterile water and physiological saline, vegetable oil,emulsifier, suspension agent, surfactant, stabilizer, flavoringexcipient, diluent, vehicle, preservative, binder, followed by mixing ina unit dose form required for generally accepted pharmaceuticalpractices. The amount of active ingredient included in thepharmaceutical preparations is such that a suitable dose within thedesignated range is provided.

The sterile composition for injection can be formulated in accordancewith conventional pharmaceutical practices using distilled water forinjection as a vehicle. For example, physiological saline or an isotonicsolution containing glucose and other supplements such as D-sorbitol,D-mannose, D-mannitol, and sodium chloride may be used as an aqueoussolution for injection, optionally in combination with a suitablesolubilizing agent, for example, alcohol such as ethanol and polyalcoholsuch as propylene glycol or polyethylene glycol, and a nonionicsurfactant such as polysorbate 80™, HCO-50 and the like.

Nonlimiting examples of oily liquid include sesame oil and soybean oil,and it may be combined with benzyl benzoate or benzyl alcohol as asolubilizing agent. Other items that may be included are a buffer suchas a phosphate buffer, or sodium acetate buffer, a soothing agent suchas procaine hydrochloride, a stabilizer such as benzyl alcohol orphenol, and an antioxidant. The formulated injection can be packaged ina suitable ampule.

In some instances, the level of sialylated glycans (e.g., branchedglycans that are sialylated on an α1,3 arm of the branched glycan in theFc region, e.g., with a NeuAc-α2,6-Gal terminal linkage) in apreparation of antibodies or Fc-containing polypeptides, produced usinga method described herein can be compared to a predetermined level(e.g., a corresponding level in a reference standard), e.g., to make adecision regarding the composition of the polypeptide preparation, e.g.,a decision to classify, select, accept or discard, release or withhold,process into a drug product, ship, move to a different location,formulate, label, package, release into commerce, or sell or offer forsale the polypeptide, e.g., a recombinant antibody. In other instances,the decision can be to accept, modify or reject a production parameteror parameters used to make the polypeptide, e.g., an antibody.Particular, nonlimiting examples of reference standards include acontrol level (e.g., a polypeptide produced by a different method) or arange or value in a product specification (e.g., an FDA label orPhysician's Insert) or quality criterion for a pharmaceuticalpreparation containing the polypeptide preparation.

In some instances, methods (i.e., evaluation, identification, andproduction methods) include taking action (e.g., physical action) inresponse to the methods disclosed herein. For example, a polypeptidepreparation is classified, selected, accepted or discarded, released orwithheld, processed into a drug product, shipped, moved to a differentlocation, formulated, labeled, packaged, released into commerce, or soldor offered for sale, depending on whether the preselected or targetvalue is met. In some instances, processing may include formulating(e.g., combining with pharmaceutical excipients), packaging (e.g., in asyringe or vial), labeling, or shipping at least a portion of thepolypeptide preparation. In some instances, processing includesformulating (e.g., combining with pharmaceutical excipients), packaging(e.g., in a syringe or vial), and labeling at least a portion of thepreparation as a drug product described herein. Processing can includedirecting and/or contracting another party to process as describedherein.

In some instances, a biological activity of a polypeptide preparation(e.g., an antibody preparation) is assessed. Biological activity of thepreparation can be analyzed by any known method. In some embodiments, abinding activity of a polypeptide is assessed (e.g., binding to areceptor). In some embodiments, a therapeutic activity of a polypeptideis assessed (e.g., an activity of a polypeptide in decreasing severityor symptom of a disease or condition, or in delaying appearance of asymptom of a disease or condition). In some embodiments, a pharmacologicactivity of a polypeptide is assessed (e.g., bioavailability,pharmacokinetics, pharmacodynamics). For methods of analyzingbioavailability, pharmacokinetics, and pharmacodynamics of glycoproteintherapeutics, see, e.g., Weiner et al., J. Pharm. Biomed. Anal.15(5):571-9, 1997; Srinivas et al., J. Pharm. Sci. 85(1):1-4, 1996; andSrinivas et al., Pharm. Res. 14(7):911-6, 1997.

The particular biological activity or therapeutic activity that can betested will vary depending on the particular polypeptide (e.g.,antibody). The potential adverse activity or toxicity (e.g., propensityto cause hypertension, allergic reactions, thrombotic events, seizures,or other adverse events) of polypeptide preparations can be analyzed byany available method. In some embodiments, immunogenicity of apolypeptide preparation is assessed, e.g., by determining whether thepreparation elicits an antibody response in a subject.

Route of administration can be parenteral, for example, administrationby injection, transnasal administration, transpulmonary administration,or transcutaneous administration. Administration can be systemic orlocal by intravenous injection, intramuscular injection, intraperitonealinjection, subcutaneous injection.

A suitable means of administration can be selected based on the age andcondition of the patient. A single dose of the pharmaceuticalcomposition containing a modified glycoprotein can be selected from arange of 0.001 to 1000 mg/kg of body weight. On the other hand, a dosecan be selected in the range of 0.001 to 100000 mg/body weight, but thepresent disclosure is not limited to such ranges. The dose and method ofadministration varies depending on the weight, age, condition, and thelike of the patient, and can be suitably selected as needed by thoseskilled in the art.

All publications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Inaddition, the materials, methods, and examples are illustrative only andnot intended to be limiting. Unless otherwise defined, all technical andscientific terms used herein have the same meaning as commonlyunderstood by one of ordinary skill in the art to which this inventionbelongs. Although methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of the presentinvention, suitable methods and materials are described herein.

EXAMPLES Example 1—Sialylation of Fc Molecules

Fc molecules were obtained or produced from various sources, glycancompositions were characterized, and anti-inflammatory activities weredetermined. The Fc molecules were tested for their ability to protectmice from joint inflammation in a mouse arthritis model using a methoddescribed in Anthony, Proc. Natl. Acad. Sci. U.S.A. 105:19571-19578(2008).

Fc molecules were derived from IVIG as follows. Commercial grade IVIGwas buffer exchanged in to phosphate buffered saline (PBS) from itsformulation buffer. This buffer exchanged IVIG was digested by papain at37° C. using 5 μg papain/mg of IVIG, and the digestion was quenched withiodoacetamide. The undigested IgG and Fc/Fab monomers were separated bysize exclusion chromatography. The Fc/Fab peak was further purified on aProtein A column to remove the Fab fragments. The purified Fc wasconcentrated before performing the sialylation reaction.

Sialylation of the Fc or IVIG substrate was performed as follows. Thesubstrate (75 mg/mL) was incubated at 37° C. for 24-48 hours with 50 mMUDP-galactose and 20 mU of bovine milk beta-1,4-galactosyltransferaseper mg of substrate. The galactosylated substrate was further incubatedat 37° C. for 48-72 hours with 80 mM CMP-sialic Acid and the specifiednumber of units of alpha-2,6-sialyltransferase per mg of substrate forsialylation. Enzyme activity was determined as described in Anumula,Glycobiol. 22:912-917 (2012).

In another method, Fc was recombinantly expressed in and purified fromCHO cells, and was subsequently sialylated using a recombinantsialyltransferase enzyme. The glycoprotein contained branched glycanshaving higher levels of sialic acid on the α1-3 arm of the branchedglycans with a NeuAc-α2,6-Gal terminal linkage, relative to thereference glycoprotein. As depicted in FIG. 4 (top panel), Fcrecombinantly expressed in CHO cells contained sialic acids linked togalactose in α2,3 linkage that were attached to both the α1,3 and α1,6arms of the branched glycans.

Interestingly, Fc that was derived from IVIG and sialylated using humanST6Gal sialyltransferase enzyme (expressed in and purified from E. colicells, 6.5 mU enzyme/mg of substrate) contained sialic acids linked togalactose in α2,6 linkage that were preferentially attached to the α1,6arms of the branched glycans (FIG. 4, middle panel). When assayed in themouse model of inflammation, these Fc molecules did not exhibitanti-inflammatory activity.

Surprisingly, when Fc that was derived from IVIG and sialylated usinghuman ST6Gal sialyltransferase enzyme (expressed in and purified fromCHO cells, 0.26 mU enzyme/mg of substrate), the Fcs contained sialicacids were linked to galactose in α2,6 linkage that were preferentiallyattached to the α1,3 arms of the branched glycans (FIG. 4, bottompanel). When assayed in the mouse model of inflammation, these Fcmolecules exhibited anti-inflammatory activity.

In another exemplary method, a preparation of IVIG was obtained, andglycan composition was determined. About 5% to about 20% of the totalglycans in the IVIG preparation contained one sialic acid on each glycan(i.e., were monosialylated). Further, greater than about 90% of thesemonosialylated glycans contained a sialic acid on an α1,3 arm of thebranched glycans with a NeuAc-α2,6-Gal terminal linkage.

In another method, Fc molecules from IVIG were sialylated with humanST6Gal sialyltransferase (recombinantly expressed in and purified frominsect cells, 0.42 mU enzyme/mg of substrate). The sialyltransferasepreferentially sialylated the α1,3 arms of the branched glycans. TheseFc molecules exhibit anti-inflammatory activity in the mouse model ofinflammation.

1. A method of producing a pharmaceutical preparation comprisingglycoproteins comprising an Fc region, wherein the branched glycans onthe Fc region are selectively sialylated on the α1-3 arm at apredetermined level comprising: contacting a sialyltransferase enzymewith a preparation comprising glycoproteins comprising an IgG Fc regionunder conditions suitable for sialylation of a plurality of saidbranched glycans by the enzyme; measuring the level of branched glycanshaving a sialic acid on said α1-3 arm and/or on the α1-6 arm; processingsaid preparation into a pharmaceutical preparation if said level isequivalent to said predetermined level; thereby producing apharmaceutical preparation comprising glycoproteins comprising an Fcregion, wherein the branched glycans on the Fc region are selectivelysialylated on the α1-3 arm at a predetermined level.
 2. The method ofclaim 1, wherein said predetermined level is at least 95% of branchedglycans having a sialic acid on said α1-3 arm.
 3. (canceled)
 4. Themethod of claim 1, wherein said sialyltransferase enzyme is a ST6Gal-Ienzyme.
 5. The method of claim 1, wherein said α1,3 arm of the branchedglycans are sialylated with a NeuAc-α2,6-Gal terminal linkage.
 6. Amethod of increasing anti-inflammatory activity of a referenceglycoprotein preparation, comprising: providing a reference glycoproteinpreparation comprising glycoproteins comprising an IgG Fc region; andsialylating the branched glycans on the Fc region on the α1-3 arm of aplurality of said branched glycans to produce a sialylated glycoproteinpreparation; wherein said glycoproteins in said reference glycoproteinpreparation are not IgG glycoproteins or do not consist essentially ofan Fc region derived from IgG glycoproteins; and wherein said sialylatedglycoprotein preparation has an increased level of anti-inflammatoryactivity relative to the level of anti-inflammatory activity of saidreference glycoprotein preparation.
 7. The method of claim 6, furthercomprising measuring in said sialylated glycoprotein preparation thelevel of said branched glycans having a sialic acid on the α1-3 armand/or measuring the level of said branched glycans having a sialic acidon the α1-6 arm.
 8. The method of claim 1, further comprising processingsaid sialylated glycoprotein preparation into a pharmaceuticalpreparation if the level of branched glycans having a sialic acid on theα1-3 arm and/or the level of branched glycans having a sialic acid onthe α1-6 arm meets a predetermined level.
 9. The method of claim 8,wherein said the predetermined level is at least about 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%of the branched glycans having a sialic acid on the α1,3 arm.
 10. Themethod of claim 8, wherein said predetermined level is less than about40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or less of the branched glycanshaving a sialic acid on the α1,6 arm.
 11. A method of increasinganti-inflammatory activity of a reference glycoprotein preparation,comprising: providing a reference glycoprotein preparation comprisingglycoproteins comprising an IgG Fc region; and sialylating the branchedglycans on the Fc region on the α1-3 arm of a plurality of said branchedglycans to produce a sialylated glycoprotein preparation; measuring insaid sialylated glycoprotein preparation the level of said branchedglycans having a sialic acid on the α1-3 arm and/or measuring the levelof said branched glycans having a sialic acid on the α1-6 arm; andprocessing said sialylated glycoprotein preparation into apharmaceutical preparation if the level of branched glycans having asialic acid on the α1-3 arm and/or the level of branched glycans havinga sialic acid on the α1-6 arm meets a predetermined level; wherein saidsialylated glycoprotein preparation has an increased level ofanti-inflammatory activity relative to the level of anti-inflammatoryactivity of said reference glycoprotein preparation.
 12. The method ofclaim 11, wherein said predetermined level of branched glycans having asialic acid on the α1-3 arm is at least 95% and said predetermined levelof branched glycans having a sialic acid on the α1-6 arm is less than5%.
 13. (canceled)
 14. (canceled)
 15. A method of manufacturing apharmaceutical product comprising glycoproteins comprising an IgG Fcregion comprising: providing a preparation comprising glycoproteinscomprising an IgG Fc region; measuring the level of branched glycans onthe Fc region in said preparation having a sialic acid on the α1-3 armand/or on the α1-6 arm; and processing the preparation into apharmaceutical product if the level of said branched glycans having asialic acid on the α1-3 arm and/or on the α1-6 arm is equivalent to apredetermined level, thereby manufacturing a pharmaceutical productcomprising glycoproteins comprising an IgG Fc region.
 16. The method ofclaim 15, wherein the predetermined level is a pharmaceuticalspecification of greater than 25% branched glycans having a sialic acidon the α1-3 arm and/or less than 40% branched glycans having a sialicacid on the α1-6 arm. 17.-23. (canceled)
 24. The method of claim 1,wherein said glycoproteins are derived from plasma.
 25. The method ofclaim 1, wherein said glycoproteins are recombinant glycoproteins. 26.(canceled)
 27. (canceled)
 28. A pharmaceutical preparation comprisingglycoproteins comprising an Fc region, wherein at least 95% of branchedglycans on the Fc region have a sialic acid on the α1-3 arm and do nothave a sialic acid on the α1-6 arm, and wherein said preparation hasanti-inflammatory activity.
 29. (canceled)
 30. A pharmaceuticalpreparation comprising a plurality of glycoproteins comprising an IgG Fcregion, wherein the IgG Fc region of each of the plurality ofglycoproteins comprises a first branched glycan sialylated on the α1-3arm, and wherein said pharmaceutical preparation has anti-inflammatoryactivity.
 31. The pharmaceutical preparation of claim 30, wherein saidIgG Fc region of the plurality of glycoproteins further comprises asecond branched glycan.
 32. The pharmaceutical preparation of claim 30,said IgG Fc region of the plurality of glycoproteins further comprises ahigh mannose glycan.
 33. The pharmaceutical preparation of claim 30,said IgG Fc region of the plurality of glycoproteins further comprises asecond branched glycan sialylated on the α1-3 arm or on the α1-6 arm.34.-44. (canceled)